Project Summary/Abstract This goal of this proposal is to advance our understanding of the mechanisms by which tyrosine recombinases recognize and assemble on their target DNA sequences and achieve coordinated pairwise cleavage of DNA strands to reach a recombined product. The proposal is significant because tyrosine recombinases are widely used genome editing tools, but their potential for improving human health is currently hindered by lack of understanding of their mechanisms for site selection and for control over activity and recombination direction (i.e., integration versus excision). Innovation in this proposal arises from the application of solution NMR to address mechanistic knowledge gaps left unanswered by the many high resolution crystal structures of tyrosine recombinases in tetrameric complexes with DNA. Those structures have provided crisp snapshots of some of the important intermediates in the recombination pathway, but provide limited insight into the intermediates that precede them, or into the mechanisms that interconvert them. Our approach combines powerful protein- and DNA-engineering with sophisticated isotope labeling and NMR methods to characterize dynamics that enable interconversion of key intermediates in site-specific DNA recombination, focusing on those leading to site-specific assembly of the tetrameric synaptic complex, allosteric control over DNA cleavage, and isomerization of the Holliday junction (HJ) intermediate. To understand the conformational changes in both protein and DNA that accompany site selection, dimer assembly, tetramer synapsis and protomer activation, we will use solution NMR spectroscopy to: (1) determine the solution structure of Cre recombinase alone, and bound in pre-synapsed complexes with loxP DNA; (2) determine the role of protein dynamics in activating Cre for DNA cleavage by measuring dynamics in Cre and pre-synaptic complexes with DNA; (3) study how DNA intrinsic dynamics affects Cre recognition, synaptic assembly, control over Cre activity, and direction of recombination; (4) characterize the dynamic and allosteric communication pathways that enable isomerization of the central HJ for progression through the recombination reaction. To enable the NMR experiments on large homo-oligomeric complexes, we will leverage an arsenal of reagents and techniques for selective labeling of protein and DNA molecules, and for assembly of chimeric Cre-DNA complexes; together with uniform deuteration and TROSY methods, the simplified NMR spectra will facilitate resonance assignments and quantitative relaxation measurements. The proposed studies will advance our understanding of the role of dynamics in DNA recombination, and in DNA binding and remodeling enzymes in general. This knowledge could broadly impact biotechnology and its biomedical applications by facilitating the design of Cre variants with defined DNA sequence specificity and improved efficiency, and suggest new avenues for controlling its activity.